Parasitic helminth infections in animals, including humans, are typically treated by chemical drugs, because there are essentially no efficacious vaccines available. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to heartworm are typically treated monthly to maintain protective drug levels. Repeated administration of drugs to treat parasitic helminth infections, however, often leads to the development of resistant strains that no longer respond to treatment. Furthermore, many of the chemical drugs are harmful to the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater.
It is particularly difficult to develop vaccines against parasitic helminth infections both because of the complexity of the parasite's life cycle and because, while administration of parasites or parasite antigens can lead to the production of a significant antibody response, the immune response is typically not sufficient to protect the animal against infection.
As for most parasites, the life cycle of D. immitis, the helminth that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. Adult forms of the parasite are quite large and preferentially inhabit the heart and pulmonary arteries of an animal. Males worms are typically about 12 cm (centimeters) to about 20 cm long and about 0.7 mm to about 0.9 mm wide; female worms are about 25 cm to about 31 cm long and about 1.0 to about 1.3 mm wide. Sexually mature adults, after mating, produce microfilariae which are only about 300 .mu.m (micrometers) long and about 7 .mu.m wide. The microfilariae traverse capillary beds and circulate in the vascular system of dogs in concentrations of about 10.sup.3 to about 10.sup.5 microfilariae per ml of blood. One method of demonstrating infection in dogs is to detect the circulating microfilariae.
If dogs are maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by female mosquitos during blood feeding on an infected dog, subsequent development of the microfilariae into larvae occurs in the mosquito. The microfilariae go through two larval stages (L1 and L2) and finally become mature third stage larvae (L3) of about 1.1 mm length, which can then be transmitted back to a dog through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood. "Occult" infection with heartworm in dogs is defined as that wherein no microfilariae can be detected, but the existence of adult heartworms can be determined through thoracic examination.
Heartworm not only is a major problem in dogs, which typically cannot even develop immunity upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy), but is also becoming increasingly widespread in other companion animals, such as cats and ferrets. Heartworm infections have also been reported in humans. Other parasitic helminthic infections are also widespread, and all require better treatment, including a preventative vaccine program.
One method by which parasites evade a host animal's immune system appears to be by neutralizing at least a portion of the immune response mounted by the host animal. Articles in the literature speculate that by producing an enzyme such as glutathione peroxidase, glutathione transferase and/or superoxide dismutase, parasites may resist the effects of oxidants produced by the host cellular immune system in response to infection. Cookson et al., 1992, Proc. Natl. Acad. Sci. 89, 5837-5841, for example, speculate that soluble forms of Brugia glutathione peroxidase may inhibit the oxidative burst of leukocytes (burst of hydrogen peroxide) and neutralize secondary products of lipid peroxidation, thus providing a possible explanation of why these parasites are apparently resistant to immune effector mechanisms of immune-mediated cytotoxicity.
Glutathione peroxidase has been identified in, and the gene encoding the enzyme has been cloned from, a number of organisms, including the filariid parasites Brugia pahangi, Brugia malayi and Wuchereria bancrofti as well as the trematode Schistosoma mansoni. The nucleotide sequence of the genes encoding B. malayi and B. pahangi glutathione peroxidases were reported to be 93.7% identical, whereas the nucleotide sequence of the genes encoding W. bancrofti and B. pahangi glutathione peroxidases were reported to be 83.1% identical; see, for example, Cookson et al., 1993, Mol. Biochem. Parasitol. 58, 155-160. Surface-labelling and antibody studies suggest that in Brugia, glutathione peroxidase appears to be present on L4 and adult parasites, but not on L3 parasites; see, for example, Selkirk et al., 1990, Mol. Biochem. Parasitol. 42, 31-43. A soluble form of glutathione peroxidase also appears to be secreted from such parasites.
Although glutathione peroxidase is a major glycoprotein of Brugia, glutathione peroxidase production in D. immitis is undetectable according to Callahan et al., 1991, Mol. Biochem. Parasitol. 49, 245-252. Callahan et al., ibid., suggest that D. immitis uses superoxide dismutase to protect itself from cell-mediated immunity. A review article by Selkirk et al., 1992, Immunobiology 184, 263-281, discloses that one drawback with glutathione peroxidase is that the protein is not observed until post-infective L3 and that, as such, a vaccine against Gp29 may not neutralize invasive larvae and may promote lymphatic pathology.